User'S Manual

USER’S MANUAL
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For Free (Physiological) Amino Acid Analysis by GC-MS
Free (Physiological)
Amino Acid Analysis
by GC-MS
USER’S MANUAL
For Part Number KG0-7166
Phenomenex Ltd. Deutschland

Zeppelinstr. 5 411 Madrid Ave
63741 Aschaffenburg Torrance, CA 90501-1430
Deutschland USA
3882_L2
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User’s Manual
The following is a description of the symbols used in the EZ:faast manuals,

on EZ:faast packaging, and on EZ:faast kit components.
Symbol for “In Vitro Diagnostic Medical Device”
Symbol for “Manufacturer”
Symbol for “Authorised Representative In

The European Community”
Symbol for “Use By” and/or “Expiration Date”
Symbol for “Batch Code” and/or “Lot Number”
Symbol for “Catalogue Number”
Symbol for “Serial Number”
Symbol for “Flammable Substances”
Symbol for “Irritating or Harmful Substances”
Symbol for “Corrosive Substances”
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User’s Manual
TABLE OF CONTENTS
Kit Components ..........................................1

Overview ....................................................2
Sample Preparation Procedure....................4
Gas Chromatographic Analysis ....................8
Troubleshooting ........................................11
Sample Storage and Stability ....................12
Cleaning and Care of Supplies ..................12
Quality Assurance .....................................13
Product Limitations ...................................13
Ordering Information ........................... 14-15
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User’s Manual
1.0 KIT COMPONENTS

1.1 Reagents
Reagent Ingredients Volume
Reagent 1 Norvaline 0. 2 mM
Internal Standard Solution N-propanol 10% 50mL
Reagent 2
Washing Solution N-propanol 90mL
Reagent 3A
Eluting Medium Component I Sodium Hydroxide 60mL
Reagent 3B
Eluting Medium Component II N-propanol 40mL
Regent 4
Organic Solution I Chloroform 4 vials, 6mL each
Reagent 5
Organic Solution II Iso-octane 50mL
Reagent 6 Iso-octane 80%
Re-dissolution Solvent Chloroform 20% 50mL
SD 1, 2, & 3 Please refer to section 4.6
Amino Acid Standard Mixtures in the manual 2 vials of each SD, 2mL each
1.2 Supplies
Sorbent tips in racks ............................................................................................. 4x96
Sample preparation vials ....................................................................................... 4x100
Microdispenser, 20-100µL ..................................................................................... 1
Syringe, 0.6mL ......................................................................................................10
Syringe, 1.5mL ......................................................................................................10
ZB-AAA 10m x 0.25mm Amino Acid Analysis GC Column ....................................... 1
Autosampler vials with inserts ............................................................................... 4x100
FocusLiners™ ........................................................................................................ 5
EZ:faast Demo Video and Reference CD ................................................................. 1
User Manual .......................................................................................................... 1
1.3 Materials Required but Not Supplied In Kit
• 100µL-1mL pipette (SoftGrip™ pipette [Phenomenex P/N AH0-5968] or equivalent)
• 30-300µL pipette (SoftGrip™ pipette [Phenomenex P/N AH0-5967] or equivalent)
• 10-100µL pipette (SoftGrip™ pipette [Phenomenex P/N AH0-5966] or equivalent)
• Pipette tips (Phenex™ [Phenomenex P/N AH0-5917 (200µL) and AH0-5920 (1mL)]
or equivalent)
• Vortex
• Vials of an appropriate volume, with caps (see section 3.2)
• Pasteur pipettes for sample transfer (see section 3.3 step 13)
• Container for proper waste disposal
• Septa (Auto-Sep T™ 11mm [SGE P/N 041883: fits Agilent or Carlo Erba instruments]
or equivalent)
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User’s Manual
2.0 OVERVIEW
2.1 Overview
The EZ:faast amino acid analysis procedure consists of a solid phase extraction step
followed by a derivatization and a liquid/liquid extraction; derivatized samples are quickly
analyzed by gas chromatography-mass spectrometry. The solid phase extraction is performed
via a sorbent packed tip that binds amino acids while allowing interfering compounds to flow
through. Amino acids on sorbent are then extruded into the sample vial and quickly derivatized
with reagent at room temperature in aqueous solution. Derivatized amino acids concomitantly
migrate to the organic layer for additional separation from interfering compounds. Organic
layer is then removed, evaporated, and re-suspended in re-dissolution solvent and analyzed
on a GC/MS system. Total sample preparation time takes around 8 minutes and analysis is
performed in around 7 minutes for a total start to finish time of around 15 minutes.
A video included with this kit demonstrates the simplicity of the procedure. Please be
aware that some sample preparation steps described in the video may be different than what
is described in this users manual. Please use the video as a general guide, but follow the exact
steps and sequence described in this manual.
2.2 Amino Acids and Related Compounds in Physiological Samples

The EZ:faast method has been developed for the analysis of more than 60 aliphatic and
aromatic amino acids, including primary and secondary amines. Further amino acids and
related compounds may be analyzed with this kit. A brief adjustment of gas chromatographic
conditions may be necessary. Please contact Phenomenex for method modifications and other
LC and GC amino acid kits. Included with the EZ:faast kit is a CD that contains spectral librar-
ies for most amino acid derivatives as run by the EZ:faast methodology. Spectral libraries are
included for Varian Saturn™ and Agilent Chemstation software systems.
Table 1 - Comprehensive list of amino acids and related compounds prepared by EZ:faast for
GC/MS analysis (internal standard listed in bold).
GC/MS Major Ions Observed (SIM) LOD* (nmol/mL)

Alter. Instrument S/N 3:1
Chemical Name Abbreviation Abb. Agilent 5973 Varian Saturn 2000 FID MS
Ethanolamine 116, 117
Alanine ALA A 130, 88 130, 70 1 0.1
Alliin 216, 173, 129
Sarcosine SAR 130, 217 130, 88 1 0.1
Glycine GLY G 116, 207 116, 102 2 0.1
α-Aminobutyric acid ABA 144, 102 144, 102 1 0.2
Valine VAL V 158, 116 116, 98, 158 0.6 0.2
Fluoro-alanine
ß-Alanine ß-ALA 129, 158, 98
ß-Aminoisobutyric acid ß-AiB 158, 116 130, 84, 144 4 0.2
ß-Amino-n-butyric acid ßABA 88, 70
Norvaline NORV 158, 72 158, 116
Leucine LEU L 172, 86 172, 130 0.7 0.1
allo-Isoleucine aILE 172, 130 130, 101 0.7 0.1
Isoleucine ILE I 172, 130 130, 101 0.7 0.2
Homoserine HSER 102, 128, 143
Norleucine NLE 172, 86
Threonine THR T 160, 101 101 2 0.2
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User’s Manual
Table 1 - (continued)
γ-Amino-n-butyric acid** GABA 130, 144, 172
Serine SER S 146, 203 101, 86 2 0.2
Proline PRO P 156, 243 156, 114 1 0.1
Asparagine ASN N 155, 69 113 2 2.5
3-Methyl-cysteine 172,259,130
Pipecolic Acid HPRO
Thioproline TPR 174, 147 174, 147 0.4 0.1
Aspartic acid ASP D 216, 130 216, 130 0.9 0.1
Methionine MET M 203, 277 101, 203, 129 0.9 0.2
3-Hydroxyproline 3HYP 172,259,130
4-Hydroxyproline 4HYP OHPro 172, 86 172, 86 2 0.2
Phenyl-glycine PHE-GLY
Seleno-methionine Se-MET
Glutamic acid GLU E 230, 170 84, 142 2 0.2
Phenylalanine PHE F 206, 190 147, 128, 91 0.5 0.2
α-Aminoadipic acid AAA 244, 98 98, 125 1 0.2
Cysteine CYS C 248, 162, 206
4-Aminobenzoic acid PABA 265, 206, 163
Homophenylalanine HPHE
α-Aminopimelic acid APA 198, 258, 286 198, 138, 112 0.5 0.4
Chloro-phenylalanine Cl-PHE
Histamine HA 180, 168, 223
Glutamine GLN Q 84, 187 84, 112 8 10
Theanine THE 112, 215
Bicine 290,260
2,4-Diamino-n-butyric acid DABA 203, 142, 245
Glycyl-glycine (dipeptide) GLY-GLY 117, 144, 201
Homocysteine HCYS 142, 203
Methionine sulfone
Methionine sulfoxide 229,182,138
S-Carboxymethyl-cysteine 144,203,262
Ornithine ORN O 156, 70 156, 139, 114 1 0.2
Glycyl-proline (dipeptide) GPR G-P 70, 300 153, 114 1 5
Tyramine 120,107,162
Lysine LYS K 170, 128 153, 170, 128 1 0.2
Threonine-aspartic acid (dipeptide) THR-ASP 218, 360, 130
Histidine HIS H 282, 168 267, 222, 136 1 0.2
Naphthyl-alanine
Seleno-cystine Se-C-C
Hydroxylysine (2 isomers) HLY OHLys 129, 169 87, 129 2 10
Tyrosine TYR Y 206, 107 164, 107 0.4 0.2
Diaminopimelic DAPA 256, 168
Proline-hydroxyproline (dipeptide) PHP 156, 186 156, 114 0.9 10
Tryptophan TRP W 130 130 0.4 0.1
Lysine-alanine (dipeptide) LYS-ALA 170, 224, 153
Dopamine DA 179, 136, 123
3-Nitrotyrosine 152, 209
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User’s Manual
Table 1 - (continued)
Aspartame 302
Cystathionine CTH 203, 272 146, 114 4 10
3,4-Dihydroxyphenylalanine DOPA 222, 123
Cystine C-C (Cys)2 248, 216 114, 173 4 10
Serotonin SRO 146,288,348
Homocystine HC-CH (Hcys)2 230, 188, 128
Arginino succinic acid ARG-SUC 441, 326
Ethionine ETH 203, 291, 143
*LODs were determined for amino acids included in standard mixtures provided with the kit
**Several amino acids coelute under the chromatographic conditions specified in the user manual (e.g. - GABA &SER)
2.3 Storage and Stability

Store Reagents 1, 3B and 4 at 4°C (do not freeze!). Store amino acid standard solutions in
the freezer. All other components may be stored at room temperature. For your convenience, the
bottom of the reagent box has been designed as a tray that can be easily lifted from the work
station and placed in the refrigerator when the kit is not in use for an extended period of time.
All components are guaranteed for 12 months or more (see label on bottle/vial) from the date
of purchase when stored at recommended temperatures and used as described in this manual.
Please review the Instruction Manual included with the Drummond® Dialamatic Microdispenser
for recommended usage and warranty information. Please observe recommendations for solvent
bottle handling and syringe cleaning in Section 7.0 of this manual.
2.4 Safety
Although the concentration of all toxic components in any of the reagent bottles is low,
for safety reasons the sample preparation station should be placed in an exhaust hood and
protective gloves and goggles should be worn. When working with biological fluids, please
take any necessary precautions to prevent infection with blood borne pathogens. Appropriate
bio-safety precautions and disposal of bio-hazardous wastes should be followed.
3.0 SAMPLE PREPARATION PROCEDURE

3.1 Setup
The EZ:faast kit packaging has been designed as an efficient workstation. It holds a
reagent tray, a vial rack, a pipette rack and a section for sorbent tips and vials. To speed up
sample preparation it is recommend that the workstation be arranged as shown in figure 1a.
By following directions and markings on the reagent box by breaking it along perforations it
can be transformed into a reagent tray. When the kit is not in use for several days, the reagent
tray (figure 1b) may be conveniently removed and placed in the refrigerator.
3.2 Preparing the Eluting Medium

The volume of prepared Eluting Medium depends upon the number of samples to be ana-
lyzed during the day (200µL/sample). The eluting medium should be prepared fresh each day:
1. Use capped vials of appropriate size (not included) for preparation of the Eluting Medium
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User’s Manual
2. Combine 3 parts Reagent 3A (Eluting Medium Component I) with 2 parts Reagent 3B (Eluting
Medium Component II) in an appropriate sized vial (see Table 2, page 5, for reagent volumes
based on number of samples). Mix briefly.
3. Store prepared eluting medium during the day at room temperature. Discard any unused mixture
at the end of the day.
WORKSTATION ARRANGEMENT - (FIGURE 1)

To speed up sample preparation it is recommended that the workstation be arranged as
shown below.
Figure 1a Figure 1b
Table 2 - For your convenience check the table below to determine the volume of Eluting Medium
components needed depending on your number of samples:
Number of Reagent 3A Eluting Reagent 3B Eluting
Samples Medium Component I Medium Component II
2 300µL 200µL
4 600µL 400µL
7 900µL 600µL
12 1.5mL 1.0mL
14 1.8mL 1.2mL
19 2.4mL 1.6mL
24 3.0mL 2.0mL
29 3.6mL 2.4mL
34 4.2mL 2.8mL
39 4.8mL 3.2mL
44 5.4mL 3.6mL
49 6.0mL 4.0mL
3.3 Sample Preparation by SPE and Derivatization

Prepare Eluting Medium first; refer to section 3.2 for preparation protocol. The freshly
prepared Eluting Medium vial may be placed in one of the empty slots in the reagent tray.
1. For each sample, line up one glass sample preparation vial in the vial rack (Figure 2). Be aware
of some variability in vial opening and sorbent tip dimensions, which may prevent the tip from
reaching to the bottom of the sample preparation vial.
Note: Droplets of liquid in SPE tip or spilled sorbent particles will not affect the precision of the assay in any way.
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User’s Manual
GLASS VIAL LINE UP - (FIGURE 2)

For each sample, line up one glass sample preparation vial in the vial rack.
Figure 2
2. Pipette 100µL sample (serum, plasma, urine or other), and 100µL Reagent 1 (Internal Standard
Solution) into each sample preparation vial.
Caution: The pH of biological samples is usually around 7. After the addition of Reagent 1 (Internal
Standard) the mixture has the correct pH for successful loading onto the SPE tip as described in the
next step. With other samples make sure that the sample + Reagent 1 mixture has a pH between pH
1.5 and pH 6.0!
Note: Samples with amino acid concentrations higher than 10mmol/L (10µmol/mL; e.g. dark colored urine) should
be analyzed by pipetting only 50µL (or 25µL) sample in the sample preparation vial instead of 100µL. Concen-
trations recorded as a result of the GC analysis will be half (one quarter) of the actual concentrations for these
samples. Conversely, when low concentrations of amino acids have to be quantified, the volume of sample to be
prepared should be 200µL or more. The total amount of amino acids present in the sample to be loaded onto
the SPE tip should not exceed 1.2 µmols.
3. Attach a sorbent tip to a 1.5mL syringe and loosen the syringe piston; immerse the tip and let
the solution in the sample preparation vial pass through the sorbent tip by SLOWLY pulling back
the syringe piston, in SMALL steps.
Caution: Do not quickly pull back the piston. Try to take at least one minute to pass low viscosity
sample (such as urine or standard) through the sorbent tip. For very viscous samples like concen-
trated plasma, up to 200µL of water can be added to ease the sample transfer through the sorbent.
The syringe should be capable of drawing all sample, and subsequent wash reagent into the barrel.
Watch as the liquid accumulates inside the syringe barrel and move the piston only as the accumu-
lation slows down. Urine passes relatively fast through the sorbent bed, while serum and plasma
pass much slower. If you run out of piston range, detach the sorbent tip, expel the solution from the
syringe barrel, then reattach the sorbent tip and proceed with sample preparation.
Note: the sorbent tip should stay in the sample preparation vial through steps 3-8 (see figure 3) even while
dispensing reagents. In case the sorbent tip cannot reach to the bottom of the vial tilt the vial to about 45°, push
the tip into the vial gently, and proceed with the SPE step.
4. Pipette 200µL Reagent 2 (Washing Solution) into the same sample preparation vial. Pass the
solution SLOWLY through the sorbent tip and into the syringe barrel. Drain the liquid from the
sorbent bed by pulling air through the sorbent tip. Detach the sorbent tip, and leave it in the
sample preparation vial, then discard the liquid accumulated in the syringe.
Note: save the syringe, as it can be reused with many other samples. For convenience place it into the pipette rack.
5. Pipette 200µL Eluting Medium (prepared fresh each day, section 3.2) into the same sample
preparation vial.
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User’s Manual
KEEP THE SORBENT TIP IN THE VIAL - (FIGURE 3)

Keep the sorbent tip in the sample preparation vial through steps 3-8, even while dispens-
ing reagents.
Figure 3
6. Pull back the piston of a 0.6 mL syringe halfway up the barrel and attach the sorbent tip used in
steps 3-8.
7. Wet the sorbent with Eluting Medium; watch as the liquid rises through the sorbent particles and
stop when the liquid reaches the filter plug in the sorbent tip.
8. Eject the liquid and sorbent particles out of the tip and into the sample preparation vial. Repeat
step 7 and 8 until the sorbent particles in the tip are expelled into the sample preparation vial.
Only the filter disk should remain in the empty tip, see figure 4. Keep the syringe as it can be
reused with many other samples.
9. Using the adjustable Drummond Dialamatic Microdispenser (included) transfer 50µL Reagent 4
into the sample preparation vial.
Caution: Avoid cross-contamination by not touching the inner wall of the sample vial with the tip of
the Microdispenser. The piston will ensure proper transfer of liquids into the vial without the need of
touching the vial wall. Use the same Microdispenser with both Reagents 4 and 5. There is no need to
change Microdispenser tips or to wash between uses. Change Microdispenser tips only when broken.
Warning: Do not use regular pipettes and tips with Reagents 4 and 5 as they will contaminate the
sample! Use the included Microdispenser for Reagents 4 and 5 ONLY!
Note: for all subsequent sample preparation steps use a vortex mixer set in the touch (pulse) mode (to about 80%
of max speed) for any mixing operations.
10. Emulsify the liquid in the vial by repeatedly vortexing for about 5-8 seconds. During vortexing
hold the sample vial firmly between fingers, and keep it straight as you push it onto the vortex
plate. Do not let vial wobble, otherwise liquid may come out of the vial. Allow reactions to
proceed 1 minute or more. The emulsion will gradually separate into two layers.
Note: a longer reaction time than 1 minute at step 10 and at step 11, or later, at step 12, does not affect results.
11. Re-emulsify the liquids in the vial by vortexing again for about 5 seconds. Allow the reaction to
proceed for one additional minute or more.
12. Transfer with the Microdispenser 100µL Reagent 5 (50µL twice, for convenience) and mix for
about 5 seconds. Let the reaction proceed for one more minute.
13. Transfer part of the (upper) organic layer (about 50-100µL) using a Pasteur pipette into an
autosampler vial (included). Avoid transferring aqueous layer along with the organic layer.
Evaporate the solvent SLOWLY to almost dry under a gentle stream of nitrogen. Do not
leave samples under the nitrogen stream for more than 10 minutes! Re-dissolve amino acid
derivatives in 100µL (or less) of Reagent 6. Transfer the reconstituted sample into an insert
(included) using a Pasteur pipette, and place the insert in the same autosampler vial. The
reconstituted sample is ready for GC/MS analysis (see GC/MS set up and calibration in section 4).
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User’s Manual
3.4 Optimizing Sample Preparation Time

For experienced users, sample preparation proceeds in 7-8 minutes per sample. This process
can be further improved by preparing up to ten samples at a time. For example, at step 2 dis-
pense Reagent 1 (and at later steps all other reagents) in ten vials successively, using the same
pipette tip. At step 9, after dispensing Reagent 4, vortex 2-3 vials simultaneously. During each
one minute wait at steps 10-12, prepare autosampler vials for sample transfer.
SORBENT TIP - (FIGURE 4)

Wet the sorbent with Eluting Medium and stop before it gets to the filter then eject the
liquid and sorbent particles out of the tip.
Figure 4
Filter
Sorbent
4.0 GAS CHROMATOGRAPHIC ANALYSIS

4.1 Column For EZ:faast Physiological Amino Acid Analysis by GC-MS
The Zebron ZB-AAA GC column comes without a cage. Connect the ends of the column in
the usual manner; rest the column coil on the oven bracket. Keep the pieces of thermal thread
spaced evenly around the column coil. The maximum column temperature is 320/340°C.
Caution: Always use safety glasses while installing the GC column.
4.2 Instrument Settings:

GC
Injection* Split 1:15@250°C, 1.5-2µL (with hot needle, see section 4.5)
Carrier Gas Helium 1.1mL/min constant flow
Oven program 30°C/min from 110° to 320°C
MS
MS Source 240°C
MS Quad 180°C
Auxiliary 310°C
Scan Range: 45-450 m/z
Sampling Rate 2² (3.5 scans/s)
*When using a Shimadzu GC instrument, please increase the injector temperature to 300°C
For your convenience we have included the GC/MS methods for Shimadzu QP-2010, Varian
Saturn™ 2000 and Agilent 5973 GC/MS systems on the reference CD included with the kit. To
use these included methods: copy the method folder into the appropriate method folder in your
software and load.
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User’s Manual
4.3 Liners
Use the best deactivated liners supplied by the instrument manufacturer. Good results
were obtained with FocusLiners™ (included; Phenomenex P/N AG0-4680; fits Agilent and
Varian 1177 injectors). In general, the liner should carry a plug of silanized quartz or pesticide
grade glass wool.
4.4 Injection
• Split injection at a ratio of 1:10 to 1:20 is recommended
• Injection volumes of 1.5-2µL are optimal
Quasi-splitless injection mode will produce a 5 to 10 fold increase in sensitivity with
some instruments. In this mode, the split valve should be closed for an initial 5 to 7 seconds.
Before selecting this injection mode it should be checked experimentally that no significant
discrimination of late eluting amino acid derivatives takes place in comparison with common
split injection. Alternatively, instruments equipped with EPC/AFC can be operated with double
initial head pressure for 6-10 seconds.
4.5 Sampling
Both autosampler and manual sampling can be performed. If manual sampling is pre-
ferred, hot needle injection is recommended to prevent discrimination of components with
high boiling temperatures. With this technique the sample plug is completely drawn into the
syringe barrel, leaving the needle empty. The needle is inserted and kept in the hot injector for
about two seconds before injection.
4.6 Calibration Standards

For quantitation purposes, mixtures of amino acid standards should be prepared following
the Sample Preparation by SPE and Derivatization procedure described in this manual in Sec-
tion 3.3. Standard mixtures should be stored in the freezer as some amino acids are not stable
in solution. Three vials of different standard mixtures are included in the kit:
SD1: 23 amino acids, 200 nmoles/mL each, as follows:
AAA ASP GLY LEU PHE THR
ABA ßAIB HIS LYS PRO TYR
aILE C-C HYP MET SAR VAL
ALA GLU ILE ORN SER
SD2: Complementary amino acids not stable in acidic solution, 200 nmoles/mL each, as follows:
ASN GLN TRP
SD3: Complementary urine amino acids, 200 nmoles/ mL each, as follows:

APA CTH GPR HLY PHP TPR
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User’s Manual
A typical chromatogram of a mixture of all three amino acid standard solutions included in
this kit. Column and instrumental settings as specified in section 4.1-4.2.
Figure 5
Abundance ( CPS x 105 )
5.54
5.24
9
3.45
8
4.75
7
6.03
3.08
4.53
2.37
6
1.97
6.26
4.95
2.03 2.00
4.00
4.48
3.04
2.84
5
1.88
5.47
1.64
2.47
1.74
3.22
4 3.73
1.83
1.42
1.48
2.25
2.29
5.13
1.53
3.42
3
4.08
2
1
1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 min
Figure 5. Elution order: tR 1.42min ALA; 1.48 SAR; 1.53 GLY; 1.64 ABA; 1.74 VAL; 1.83 ßAIB; 1.88 IS = NVAL; 1.97
LEU; 2.00 aILE; 2.03 ILE; 2.25 THR; 2.29 SER; 2.37 PRO; 2.47 ASN; 2.84 TPR; 3.04 ASP; 3.08 MET; 3.22 HYP; 3.42
GLU; 3.45 PHE; 3.73 AAA; 4.00 APA; 4.08 GLN; 4.48 ORN; 4.53 GPR; 4.75 LYS; 4.95 HIS; 5.13 HLY; 5.24 TYR; 5.47
PHP; 5.54 TRP; 6.03 CTH and 6.26 C-C.
4.7 Calibration Procedure

Use the following standard amino acid mixtures and make duplicate injections of each to
generate the desired calibration:
Calibration Solution
I. 25µL of SD1 solution, plus 25µL of SD2 (and/or SD3 depending on your application), plus 100µL
Reagent 1 (calibration level I:50 nmoles/mL)
II. 50µL SD1+ 50µL SD2 (and/or SD3) + 100µL Reagent 1 (calibration level II:100 nmoles/mL)
III. 100µL SD1+ 100µL SD2 (and/or SD3) + 100µL Reagent 1 (calibration level III:200 nmoles/mL).
The concentration of the internal standard (IS= Norvaline) in the sample prepared for gas-
chromatographic analysis is 200 nmoles/mL (serum/urine/etc.).
Note: Disregard the first 3-5 injections when performing method calibration or after replacing column or liner.
These injections act as primers and mask active sites inside the liner and chromatographic column. Use subse-
quent duplicate runs for calibration.
Remember: the SD1, SD2, and SD3 vials should be placed in the freezer after use! Allow standards
to reach room temperature before use.
4.8 Calibration of analytical results

Calculations are performed by the Data Analysis portion of the software controlling the
analytical instrument (gas-chromatograph). Calculations and calibration are based on an in-
ternal standard. Results are reported in the units entered for the internal standard and analyte
levels in calibration mixtures.
Note: nmols/mL are equivalent to µmols/L
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User’s Manual
5.0 TROUBLESHOOTING
Problem Cause Solution
Decrease in peak height for some Possibly related to improper sample See sample standard storage, sec-
amino acids (components of amino storage tion 6.0
acid standard mixtures SD2 or SD3)
Decrease in peak height for basic Old eluting medium Prepare eluting medium daily based
amino acids on the number of samples to be
analyzed on that day
Decrease in peak height or missing Improper liner Use deactivated liners, see section
peaks for late eluting (HIS, C-C, 4.4. Analyze samples only after mak-
HC-CH), or polar amino acids (SER, ing priming injections.
HYP, GLN)
Decrease in peak height for early Sample too concentrated; the Use less sample for preparation, see
eluting amino acids capacity of the SPE tip exceeded section 3.3, step 2. Constantly moni-
tor the area for the IS peak.
Volatile derivatives evaporated Adjust nitrogen flow to minimal;
during removal of residual reagents stop evaporation before sample gets
with nitrogen gas completely dry
Low peak height for late eluting Carrier gas leak Check instrument for leaks (reinstall
derivatives the column and check o-ring on liner)
Peak height for IS (Norvaline) lower Pipetting error Recalibrate pipette used for dispens-
compared to other early eluting ing Reagent 1. Constantly monitor
amino acids in the standard mix area for IS.
Ghost peaks Pipette tips used for dispensing Use polypropylene pipette tips of
reagents or for transferring prepared appropriate quality (see order-
samples may be a source of con- ing info on page 14). Always use
taminant peaks the Microdispenser for dispensing
Reagents 4, 5, and 6. Use Pasteur
pipettes to transfer the reconstituted
sample into a glass insert.
Interfering peaks may result from Use the vials provided in the kit. For
extracted contaminants in plastic autosampler vials order Phenomenex
sample preparation vials P/N AH0-4610 and for inserts
AH0-4604.
Early deterioration of column Residual sample preparation Evaporate residual reagents at step
performance reagents degrade column stationary 13. Do not use samples prepared
phase as described in the EZ:faast kit for
the analysis of free amino acids by
GC-FID!
Interfering peaks, drug metabolites Physiological sample anticoagulants, Samples collected with EDTA and
like citrate or citric acid may inter- heparin anticoagulants are recom-
fere in the amino acid profile mended. Confirm peak identity based
on mass- spectrum.
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User’s Manual
6.0 SAMPLE STORAGE AND STABILITY

Some amino acids are chemically unstable in physiological fluids (e.g., progressive decline
of plasma glutamine and cystine in time), and also in standard mixtures. Keep samples and
standard mixtures in the freezer. Old amino acid standard mixtures and mixtures which have
not been stored properly should not be used for instrument calibration. Order fresh mixtures
from Phenomenex (see ordering info in Phenomenex catalog).
Samples prepared for GC-MS analysis following the procedure outlined in this manual
may be stored for several days in a freezer before analysis. For longer storage we recommend
that samples be desiccated with anhydrous sodium sulfate, vials be capped and placed in the
freezer. Since sample preparation is expeditious with this procedure we recommend analyzing
samples prepared freshly. Samples prepared during the day may be left on the autosampler
tray, at room temperature, to be analyzed during the night, or the next day.
7.0 CLEANING AND CARE OF SUPPLIES

The Drummond® Dialamatic Microdispenser should be flushed with isopropanol: acetone
(approx. 1:1) at the end of the day. Please review the Drummond Microdispenser users manual
for further care and use notes. The same organic mix is recommended as wash for both
manual syringes and autosamplers.
Plastic syringes used for sample clean-up by SPE can be cleaned by flushing with propa-
nol:water (1:2, v/v) mixture.
Always tightly cap the reagent bottles when not in use in order to avoid solvent evapora-
tion and alteration of reagent composition. Cover the racks holding sorbent tips when not in
use to prevent contamination.
12
User’s Manual
8.0 QUALITY ASSURANCE

All components of the EZ:faast Gas Chromatographic Amino Acid Analysis kit are subjected
to rigorous quality control testing. These measures help to ensure the best results. If poor
results occur, please contact your Phenomenex technical consultant or distributor.
9.0 PRODUCT LIMITATIONS

Phenomenex Analyte Specific Reagent products are not intended for clinical use. Because
they are not intended for clinical use, no claim or representation is made or intended for their
clinical use (including, but not limited to diagnostic, prognostic, therapeutic or blood banking).
It is the user’s responsibility to validate the performance of Phenomenex products for any par-
ticular use, since the performance characteristics are not established. Phenomenex products
may be used in clinical diagnostic laboratory systems after the laboratory has validated their
complete system as required by the Clinical Laboratory Improvements Amendments of 1988
(CLIA ‘88) regulation in the U.S. or equivalent in other countries.
Trademarks
EZ:faast Sorbent Tips are patented, Phenomenex, Inc. U.S. Patent 6,770,246
EZ:faast is a trademark of Phenomenex, Inc.
Phenex is a trademark of Phenomenex, Inc.
FocusLiner and Auto-Sep T are trademarks of SGE
SoftGrip is a trademark of Hamilton
Drummond is a registered trademark of the Drummond Corp.
Registered names, trademarks, etc. used in this document, even when not
specifically marked as such, are not to be considered unprotected by Law.
©
2005-2006 Phenomenex, Inc. All rights reserved.
13
ordering information
User’s Manual
EZ:faast™ Kit
Each kit includes: a ZB-AAA GC column (or EZ:faast AAA LC column), GC liners with GC
kits, sample prep and derivatization reagents, sample prep vials, AA standard mixtures,
SPE sorbent tips, vial rack, autosampler vials with inserts come with MS kits, Microdis-
penser for Reagents 4 and 5, and demo video.
Description Order No. Unit
GC-FID Free (Physiological) Amino Acid Analysis Kit KG0-7165 ea
GC-MS Free (Physiological) Amino Acid Analysis Kit KG0-7166 ea
GC-FID Protein Hydrolysate Kit KG0-7167 ea
GC-MS Protein Hydrolysate Kit KG0-7168 ea
LC/MS Free (Physiological) Amino Acid Analysis Kit with 250 x 2.0mm column KH0-7337 ea
LC/MS Protein Hydrolysate Kit with 250 x 2.0mm column KH0-7339 ea
GC Free (Physiological) Amino Acid Standards (SD1, SD2 & SD3) 2mL/vial x 2 AG0-7184 ea
GC Protein Hydrolysate Standard (SD) 2mL/vial x 2 AG0-7263 ea
LC/MS Free (Physiological) Amino Acid Standards for LC (SD1, SD2, & SD3) 2mL/vial x 2 AL0-7500 ea
LC/MS Protein Hydrolysate Standard (SD) 2mL/vial x 2 AL0-7501 ea
14
User’s Manual
Phenex™ Vials
This universal vial can be used in any autosampler that utilizes a 12 x 32mm vial. It
may be used in place of crimp top and snap ring top vials. Eliminates the need of stocking
many different style vials. The top screws down in 1/3 turn and eliminates the chore of
crimping, de-crimping and snapping caps on. Cap comes with a bonded-in septa that
eliminates septa slipping into vials. Vials and caps with bonded-in septa come in one
convenient kit pack.
Clear wide mouth vial, cap and septa kit pack with:
Rubber/PTFE septa AH0-4610 1000/pk
Silicone/PTFE septa AH0-4613 1000/pk
PTFE/Silicone/PTFE septa AH0-4616 1000/pk
Amber wide mouth vial, cap and septa kit pack with:
Rubber/PTFE septa AH0-4619 1000/pk
Clear wide mouth vial, cap with pre-slit septa:
SGE FocusLiners™
Description GC Model Dimensions ID Material* Quartz Mfr P/N Order No. Unit
No. ID x L x OD(mm) (deactivated) Wool (Y/N)
Thermo Electron 8000 series 5 x 105 x 8.0 B (y) Y 092046 AG0-4679 5/pk
(Carlo Erba)
Single Taper/
Gooseneck
Liner
Agilent 5880/5890 4 x 78.5 x 6.3 B (y) Y 092003 AG0-4680 5/pk
Technologies /6890
Single Taper/
Gooseneck
Liner
PerkinElmer Autosystem 4 x 92 x 6.2 B (y) Y 092095 AG0-4681 5/pk
Single Taper/
Gooseneck
Liner
Shimadzu 17B 3.4 x 95 x 5 B (y) Y 092068 AG0-4683 5/pk
Single Taper/
Gooseneck
Liner
Varian 1075/77 4 x 72 x 6.3 B (y) Y 092025 AG0-4684 5/pk
Single Taper/
Gooseneck
Liner
Varian 1078/79 3.4 x 54 x 5 B (y) Y 092036 AG0-4685 5/pk
Double Taper/
Gooseneck
Liner
* B=Borosilicate; Deactivated=Yes (y) or No (n)
15
User’s Manual
EZ:faast - Free (Physiological) Amino Acid

Analysis by GC-MS
QUICK REFERENCE GUIDE
Summary of Procedure:
1. For each sample line up one glass sample preparation vial in the vial rack.
2. Pipette 100µL sample (serum, plasma, urine or other; see section 3.3.2) and 100µL Reagent 1 into each
sample preparation vial.
3. Attach a sorbent tip to a 1.5mL syringe; pass the solution in the sample preparation vial through the
sorbent tip by slowly pulling back the syringe piston.
4. Pipette 200µL Reagent 2 (Washing Solution) into the sample preparation vial.
5. Slowly pass the solution through the sorbent tip and into the syringe barrel.
6. Detach the sorbent tip, and discard the liquid accumulated in the syringe.
7. Pipette 200µL Eluting Medium (prepared fresh each day, section 3.2) into the sample preparation vial.
8. Pull back the piston of a 0.6mL syringe halfway up the barrel and attach the sorbent tip.
9. Wet the sorbent with Eluting Medium; stop when the liquid reaches the filter plug in the sorbent tip.
Detach Here
10. Eject the liquid and sorbent out of the tip and into the sample preparation vial. Repeat, until all sorbent
particles in the tip are expelled into the sample preparation vial. Discard the empty tip.
11. Using the Drummond Dialamatic Microdispenser, transfer 50µL Reagent 4.
12. Emulsify by repeatedly vortexing the solution for about 5 seconds. Allow reaction to proceed for 1 minute
or more.
13. Vortex the solution again for a few seconds to re-emulsify the content of the vial. Allow the reaction to
proceed for at least one additional minute.
14. Using the Microdispenser, transfer 100µL Reagent 5, and re-emulsify by vortexing for about 5 seconds.
Let the reaction proceed for 1 minute.
15. Transfer part of the (upper) organic layer (50-100µL) using a Pasteur pipette into an autosampler vial.
Avoid transferring aqueous layer along with the organic layer. Evaporate the solvent SLOWLY to almost
dry under a gentle stream of nitrogen (max 10 min). Re-dissolve amino acid derivatives in 100µL (or less)
of Reagent 6 using a Pasteur pipette. Transfer the reconstituted sample into an insert, and place the insert
in the same autosampler vial. The reconstituted sample is ready for GC/MS analysis.
Gas Chromatographic Analysis

GC Settings
Injection Split 1:15 @ 250°C, 2.0µL
Carrier Gas Helium, 1.1mL/min
Oven Program 30°C/min from 110° to 320°C
MS Settings
MS Source 240°C
MS Quad 180°C
Auxiliary 310°C
Scan Range 45-450 m/z
Sampling Rate 2² (3.5 scans/s)
3882_L2
User’s Manual
EZ:faast™ Kit
Each kit includes: a ZB-AAA GC column (or EZ:faast AAA LC column), GC liners with GC
kits, sample prep and derivatization reagents, sample prep vials, AA standard mixtures,
SPE sorbent tips, vial rack, autosampler vials with inserts come with MS kits, Microdis-
penser for Reagents 4 and 5, and demo video.
GC-FID Free (Physiological) Amino Acid Analysis Kit KG0-7165 ea
GC-MS Free (Physiological) Amino Acid Analysis Kit KG0-7166 ea
GC-FID Protein Hydrolysate Kit KG0-7167 ea
GC-MS Protein Hydrolysate Kit KG0-7168 ea
GC Free (Physiological) Amino Acid Standards (SD1, SD2 & SD3) 2mL/vial x 2 AG0-7184 ea
GC Protein Hydrolysate Standard (SD) 2mL/vial x 2 AG0-7263 ea
LC/MS Free (Physiological) Amino Acid Standards for LC (SD1, SD2, & SD3) 2mL/vial x 2 AL0-7500 ea
LC/MS Protein Hydrolysate Standard (SD) 2mL/vial x 2 AL0-7501 ea
USER’S MANUAL
3882_L
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USER’S MANUAL

Phenomenex Ltd. Deutschland

The following is a description of the symbols used in the EZ:faast manuals,

Symbol for “In Vitro Diagnostic Medical Device”

Symbol for “Manufacturer”

Symbol for “Authorised Representative In

Symbol for “Use By” and/or “Expiration Date”

Symbol for “Batch Code” and/or “Lot Number”

Symbol for “Catalogue Number”

Symbol for “Serial Number”

Symbol for “Flammable Substances”

Symbol for “Irritating or Harmful Substances”

Symbol for “Corrosive Substances”

Kit Components ..........................................1

1.0 KIT COMPONENTS

2.2 Amino Acids and Related Compounds in Physiological Samples

GC/MS Major Ions Observed (SIM) LOD* (nmol/mL)

2.3 Storage and Stability

3.0 SAMPLE PREPARATION PROCEDURE

3.2 Preparing the Eluting Medium

WORKSTATION ARRANGEMENT - (FIGURE 1)

3.3 Sample Preparation by SPE and Derivatization

GLASS VIAL LINE UP - (FIGURE 2)

KEEP THE SORBENT TIP IN THE VIAL - (FIGURE 3)

3.4 Optimizing Sample Preparation Time

SORBENT TIP - (FIGURE 4)

4.0 GAS CHROMATOGRAPHIC ANALYSIS

4.2 Instrument Settings:

4.6 Calibration Standards

SD3: Complementary urine amino acids, 200 nmoles/ mL each, as follows:

4.7 Calibration Procedure

4.8 Calibration of analytical results

6.0 SAMPLE STORAGE AND STABILITY

7.0 CLEANING AND CARE OF SUPPLIES

8.0 QUALITY ASSURANCE

9.0 PRODUCT LIMITATIONS

* B=Borosilicate; Deactivated=Yes (y) or No (n)

EZ:faast - Free (Physiological) Amino Acid

Gas Chromatographic Analysis

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